Methods for treating diabetes

ABSTRACT

Methods for treating diabetes by administering an inhibitor of GDF-8, or a related member of Transforming Growth Factor-beta (TGF-β) superfamily of structurally-related growth factors (e.g., GDF-11) are disclosed. Also disclosed are methods for upregulating expression of hexose transporters, such as GLUT4 and GLUT1, in a subject by administering an inhibitor of GDF-8. Also disclosed are methods for increasing glucose uptake by cells in a subject, by administering an inhibitor of GDF-8.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/084,490, filed May 6, 1998, the entire contents of which arehereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] Diabetes mellitus is the most common metabolic disease worldwide.Every day, 1700 new cases of diabetes are diagnosed in the UnitedStates, and at least one-third of the 16 million Americans with diabetesare unaware of it. Diabetes is the leading cause of blindness, renalfailure, and lower limb amputations in adults and is a major risk factorfor cardiovascular disease and stroke.

[0003] Normal glucose homeostasis requires the finely tunedorchestration of insulin secretion by pancreatic beta cells in responseto subtle changes in blood glucose levels, delicately balanced withsecretion of counter-regulatory hormones such as glucagon. Type 1diabetes results from autoimmune destruction of pancreatic beta cellscausing insulin deficiency. Type 2 or noninsulin-dependent diabetesmellitus (NIDDM) accounts for >90% of cases and is characterized by atriad of (1) resistance to insulin action on glucose uptake inperipheral tissues, especially skeletal muscle and adipocytes, (2)impaired insulin action to inhibit hepatic glucose production, and (3)dysregulated insulin secretion (DeFronzo, (1997) Diabetes Rev.5:177-269). In most cases, type 2 diabetes is a polygenic disease withcomplex inheritance patterns (reviewed in Kahn et al., (1996) Annu. Rev.Med. 47:509-531).

[0004] Environmental factors, especially diet, physical activity, andage, interact with genetic predisposition to affect disease prevalence.Susceptibility to both insulin resistance and insulin secretory defectsappears to be genetically determined (Kahn, et al). Defects in insulinaction precede the overt disease and are seen in nondiabetic relativesof diabetic subjects. In spite of intense investigation, the genesresponsible for the common forms of Type 2 diabetes remain unknown.

[0005] One of the fundamental actions of insulin is to stimulate uptakeof glucose from the blood into tissues, especially muscle and fat. Thisoccurs via facilitated diffusion which is mediated by specific glucosetransporter proteins that insert into the plasma membrane of cells.GLUT4 is the most important insulin-sensitive glucose transporter inthese tissues. Insulin binds to its receptor in the plasma membrane,generating a series of signals that result in the translocation ormovement of GLUT4 transporter vesicles to the plasma membrane, where afirst clocking step, followed by fusion with the plasma membrane takesplace; after an activation or exposure step takes place, glucose entersthe cell. Studies in both animals ad humans indicate that alterations inGLUT4 expression, trafficking, and/or activity occur in adipose cellsand muscle in diabetes and other insulin-resistant states (Abel et al.,Diabetes Mellitus: A Fundamental and Clinical Text (1996) pp.530-543.)

[0006] New and innovative treatments for diabetes are clearly a priorityfor researchers in this field. The present invention provides suchinnovative treatments, taking advantage of the knowledge concerningGLUT4 Expression and activity, and expression and activity of relatedhexose transporters (e.g., GLUT1).

SUMMARY OF THE INVENTION

[0007] The present invention provides a method of treating diabetes andrelated diseases, such as obesity, by administering to a subject aninhibitor of GDF-8. Suitable inhibitors of GDF-8 which can be employedin the methods of the invention include, but are not limited to, GDF-8peptides (e.g., derived from the pro-domain), GDF-8 dominant-negativemutants, antibodies and antibody fragments which bind to GDF-8 (or thereceptor for GDF-8) and inhibit GDF-8 binding to its receptor, GDF-8receptor peptide antagonisists, antisense nucleic acids directed againstGDF-8 mRNA and anti-GDF-8 ribozymes.

[0008] In another aspect, the present invention provides a method ofincreasing GLUT4 expression in a cell (e.g., a muscle cell or a fat cellin a subject), or increasing glucose uptake by a cell, by administeringa GDF-8 inhibitor. Such methods can be used, not only to treat diabetesand related diseases, but also to treat several systemic problemsresulting from insufficient glucose metabolism, such as hyperglycemia.

[0009] The methods of the present invention also can be performed usingas targets other TGF-β growth factors which are related in structure andactivity to GDF-8, such as GDF-11. Accordingly, in another embodiment,the invention provides method of treating diabetes by administering to asubject an inhibitor of GDF-11, either alone or incombination with otherGDF inhibitors (e.g., an inhibitor of GDF-8).

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1A shows GLUT4 levels by immunostaining, with an anti-GLUT4antibody, in the pectoralis and the quadriceps from a wild-type mouseand a GDF-8 knockout mouse.

[0011]FIG. 1B shows GLUT4 levels, by immunostaining, with an anti-GLUT4antibody, in five different muscle samples, pectoralis, triceps,gastrocnemius, quadriceps, and iliocostal, in both a wild-type mouse anda GDF-8 knockout mouse.

[0012]FIG. 2 shows GLUT4 levels by immunostaining with an anti-GLUT4antibody in muscle from a control mouse, a GDF-8-dosed mouse, aninsulin-dosed mouse, and a GDF-8 plus insulin-dosed mouse.

[0013]FIG. 3 is a graph showing the correlation between increasedsystemic levels of GDF-8 in nude mice (as secreted from aGDF-8-expressing CHO cell tumor) and severe weight loss as compared tocontrol mice.

[0014]FIG. 4 is a graph showing the correlation between increasedsystemic levels of GDF-8 in nude mice (as secreted from aGDF-8-expressing CHO cell tumor) and overall body weight (Panel A),tumor weight (Panel B), pectoralis weight (Panel C), epididymal fatweight (Panel D) and gastrocnemius weight (Panel E) as compared to thesetissues from control mice containing CHO cell tumors not expressingGDF-8.

[0015]FIG. 5 is a graph comparing the size of GDF-8-secreting CHO celltumors in nude mice relative to control CHO cell tumors not expressingGDF-8. Tumor size was measured as cross sectional area.

[0016]FIG. 6 is a graph showing the correlation between increased GDF-8levels (from GDF-8 expressing CHO cell tumors) in nude mice and serumglucose levels (Panel A) and GLUT4 expression levels (Panel B) inmuscle, as compared to control mice containing CHO cell tumors notexpressing GDF-8.

[0017]FIG. 7 shows the effect of exogenously added GDF-8 on 3T3-L1adipocyte differentiation, as compared to no treatment, TNF-α treatment,and TGF-β1 treatment.

[0018]FIG. 8 is a Northern blot analysis showing reduced expression ofGLUT4 in 3T3-L1 cells treated with GDF-8, TGFβ and TNFα. Total cellularRNA for each sample was fractionated, immobilized to a membrane andhybridized with ³²p probes for GLUT4 mRNA.

[0019]FIG. 9 is a graph showing that treatment of differentiated 3T3-L1adipocytes with different doses of GDF-8 impairs the ability of thesecells to increase glucose uptake in response to insulin, thus leading todesensitization.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is based, in part, on the discovery thatGDF-8 downregulates expression of GLUT4 in tissues primarily in muscleand fat. Regulation of glucose metabolism by insulin is a key mechanismby which homeostasis is maintained in an animal. The action of insulinin the regulation of circulating glucose levels is to stimulate glucoseuptake in muscle and fat tissues. Insulin stimulates glucose uptake inthese tissues by increasing the translocation of GLUT4, theinsulin-sensitive glucose transporter, from an intracellular vesicularcompartment to the plasma membrane.

[0021] It was further discovered as part of the present invention thatGLUT4 expression in muscle and fat cells can be upregulated byinhibiting GDF-8. It was also discovered that glucose uptake by thesecells can be increased by inhibiting GDF-8. These effects can beadvantageously utilized to treat a variety of metabolic diseasesresulting from dysfunctional glucose metabolism (e.g., hyperglycemia)and/or insulin resistance.

[0022] Accordingly, in one embodiment, the present invention provides amethod for treating diabetes mellitus and related disorders, such asobesity or hyperglycemia, by administering to a subject an inhibitor ofGDF-8 in an amount sufficient to ameliorate the symptoms of the disease.Type 2 or noninsulin-dependent diabetes mellitus (NIDDM), in particular,is characterized by a triad of (1) resistance to insulin action onglucose uptake in peripheral tissues, especially skeletal muscle andadipocytes, (2) impaired insulin action to inhibit hepatic glucoseproduction, and (3) dysregulated insulin secretion (DeFronzo, (1997)Diabetes Rev. 5:177-269). Therefore, subjects suffering from type 2diabetes can be treated according to the present invention byadministration of a GDF-8 inhibitor, which increases sensitivity toinsulin and glucose uptake by cells.

[0023] Similarly, other diseases characterized by insulin dysfunction(e.g., resistance, inactivity or deficiency) and/or insufficient glucosetransport into cells also can be treated according to the presentinvention by administration of a GDF-8 inhibitor, which increasessensitivity to insulin and glucose uptake by cells.

[0024] Definitions

[0025] As used herein, the term “GDF-8 inhibitor” or “an inhibitor ofGDF-8” includes any agent capable of inhibiting GDF-8 activity,including but not limited to peptides (derived from GDF-8, GDF-11 orother unrelated sequences), dominant-negative protein mutants,peptidomimetics, antibodies or fragments thereof, ribozymes, antisenseoligonucleotides, or other small molecules which specifically inhibitthe action of GDF-8 while, preferably, leaving intact the activity ofTGF-β, Activin or other members of the TGF-β superfamily. The term “aGDF-11 inhibtor” also encompasses these classes of inhibitors and,preferably, specifically inhibits GDF-11. GDF-8 and GDF-11 arestructurally and functionally related members of the TGF-β family ofgrowth factors.

[0026] GDF-8 inhibitors used in the methods of the invention,particularly those derived from GDF-8 itself (e.g., GDF-8 peptides, suchas the pro-domain or portions thereof), preferably do not possess GDF-8activity. Such inhibitors and methods for their identification aredescribed in U.S. Ser. No. 60/116,639, entitled “Growth DifferentiationFactor Inhibitors and Uses Therefor”, incorporated by reference hereinin its entirety. For example, the inhibitory action of a GDF-8 inhibitorcan be assessed using a variety of art-recognized assays, such as aNorthern blot analysis of GDF-8 mRNA, or a Western blot analysis orimmunostaining analysis of GDF-8 protein levels, among others.Identified GDF-8 inhibitory compounds can be further evaluated,detected, cloned, sequenced, and the like, either in solution or afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence such as PCR, oligomer restriction(Saiki et al., Bio/Technology, 3:1008 (1985)), allele-specificoligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad.Sci. USA 80:278 (1983)), ligase-mediated gene detection (Landegren etal, Science 241:1077 (1988)), and the like.

[0027] As used herein, the term “GDF-8 activity” or “GDF-11 activity”includes any activity mediated by GDF-8 or GDF-11, respectively. Forexample, GDF-8 is known to inhibit fibroblast differentiation toadipocytes, iodulate the production of muscle-specific enzymes, e.g.,creatine kinase, modulate uptake glucose by cells, and stimulatemyoblast cell proliferation. Accordingly, the degree to which a GDF-8inhibitor prevents GDF-8 activity can be identified by, for example,testing for the ability of the inhibitor to block GDF-8 activity, asmeasured by the ability of GDF-8 to interfere with the differentiationprocess of 3T3-L1 pre-adipocytes (fibroblasts) to adipocytes, theability to modulate the activity of muscle-specific enzymes, e.g.,creatine kinase, the ability to modulate glucose uptake by cells, or theability to stimulate myoblast cell proliferation. The effect of theinhibitor on inhibition of insulin stimulation of GLUT4 expression andglucose uptake, can also be assessed, and may include measurementsbefore and after incubating in the presence of the compound.

[0028] As used herein, the term “modulate” refers to an increase infunction. For example, modulation of gene transcription or expressionrefers to upregulation of these functions. Modulation of proteinactivity refers to an increase in activity.

[0029] As used herein, the term “inhibit” refers to a decrease, whetherpartial or whole, in function. For example, inhibition of genetranscription or expression refers to any level of downregulation ofthese functions, including complete elimination of these functions.Modulation of protein activity refers to any decrease in activity,including complete elimination of activity.

[0030] As used herein, the term “diabetes” includes all known forms ofdiabetes, including type I and type II diabetes, as described in Abel etal., Diabetes Mellitus: A Fundamental and Clinical Text (1996)pp.530-543.

[0031] GDF-8 inhibitors of the invention are typically administered to asubject in “substantially pure” form. The term “substantially pure” asused herein refers to GDF-8 which is substantially free of otherproteins, lipids, carbohydrates, or other materials with which it isnaturally associated. One skilled in the art can purify GDF-8 usingstandard techniques for protein purification. The substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel. The purity of the GDF-8 polypeptide can also bedetermined by amino-terminal amino acid sequence analysis.

[0032] Specific details on the production of GDF-8 for testing anddeveloping inhibitors for use in the present invention are providedby-McPherron, et al., Nature 387:83 90 (1997), and U.S. Pat. No.5,827,733, both hereby incorporated by reference in their entirety.

[0033] As used herein, the term “hexose transporter” includes integralmembrane proteins of a cell able to transport a hexose sugar, such asglucose, from the exterior to the interior of the cell. Examples of suchtransporters are the GLUT1 and GLUT4 transporter proteins, among others,in muscle and fat cells.

[0034] As used herein, the term “modulation of GDF-8 activity” or“modulation of GDF-8 level” refers to a change in GDF-8 activity orlevel compared to its native state. This change may be either positive(upregulation), or negative (downregulation), but for the purposes ofthe present invention is preferably the latter.

[0035] Cells which are targeted by the methods of the present invention,such as muscle and fat cells, include isolated cells maintained inculture as well as cells within their natural context in vivo (e.g., infat tissue or muscle tissue, such as pectoralis, triceps, gastrocnemius,quadriceps, and iliocostal muscles).

[0036] The term “antisense nucleic acid” refers to a DNA or RNA moleculethat is complementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American 262:40 (1990)). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translate an mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetGDF-8 producing cell. The use of antisense methods to inhibit the invitro translation of genes is well known in the art (Marcus-Sakura,Anal. Biochem. 172:289 (1988)).

[0037] As used herein, a “ribozyme” is a nucleic acid molecule havingnuclease activity for a specific nucleic acid sequence. A ribozymespecific for GDF-8 mRNA, for example, would bind to and cleave specificregions of the GDF-8 mRNA, thereby rendering it untranslatable andresulting in lack of GDF-8 polypeptide production.

[0038] The term “dominant-negative mutant” refers to a GDF-8 proteinwhich has been mutated from its natural state and which interacts withGDF-8 or a GDF-8 gene, thereby inhibiting its production and/oractivity.

[0039] The “antibodies” of the present invention include antibodiesimmunoreactive with GDF-8 polypeptides or functional fragments thereof.Antibodies which consist essentially of pooled monoclonal antibodieswith different epitopic specificities, as well as distinct monoclonalantibody preparations are provided. Monoclonal antibodies are made fromantigen-containing fragments of the protein by methods well known tothose skilled in the art (Kohler et al, Nature 256:495(1975)). The term“antibody” as used in this invention is meant to include intactmolecules as well as fragments thereof, such as Fab and F(ab′)₂, Fv andSCA fragments which are capable of binding an epitopic determinant onGDF-8.

[0040] A “Fab fragment” consists of a monovalent antigen-bindingfragment of an antibody molecule, and can be produced by digestion of awhole antibody molecule with the enzyme papain, to yield a fragmentconsisting of an intact light chain and a portion of a heavy chain.

[0041] A “Fab′ fragment” of an antibody molecule can be obtained bytreating a whole antibody molecule with pepsin, followed by reduction,to yield a molecule consisting of an intact light chain and a portion ofa heavy chain. Two Fab′ fragments are obtained per antibody moleculetreated in this manner.

[0042] A “(Fab′)₂” of an antibody can be obtained by treating a wholeantibody molecule with the enzyme pepsin, without subsequent reduction.A (Fab′)₂ fragment is a dimer of two Fab′ fragments held together by twodisulfide bonds.

[0043] An “Fv fragment” is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

[0044] A “single chain antibody” (SCA) is a genetically engineeredsingle chain molecule containing the variable region of a light chainand the variable region of a heavy chain, linked by a suitable, flexiblepolypeptide linker.

[0045] GDF-8 and GDF-11 Inhibitors for Use in the Methods of theInvention

[0046] GDF-8 inhibitors suitable for use in the invention include, butare not limited to, peptides, including peptides derived from GDF-8(e.g., mature GDF-8 or the pro-domain of GDF-8) or non-GDF-8 peptides,GDF-8 dominant-negative mutants, antibodies and antibody fragments whichbind to GDF-8 (or the receptor for GDF-8) and inhibit GDF-8 binding toits receptor, GDF-8 receptor peptide antagonisists, antisense nucleicacids directed against GDF-8 mRNA and anti-GDF-8 ribozymes. Thus, GDF-8inhibitors can act at the message (transcription) level or at theprotein (expression or activity) level.

[0047] As used herein, the term “GDF-8” includes all known forms ofGDF-8 including but not limited to human GDF-8, bovine GDF-8, chickenGDF-8, murine GDF-8, rat GDF-8, porcine GDF-8, ovine GDF-8, turkeyGDF-8, and baboon GDF-8. These molecules are described in McPherron A.C. et al. (1997) Proc. Natl. Acad. Sci. 94:12457-12461, the contents ofwhich are incorporated herein by reference. The amino acid sequences forthese proteins are shown in FIG. 12.

[0048] As used herein, the term “GDF-11” includes all known forms ofGDF-11 including but not limited to human GDF-11, bovine GDF-11, chickenGDF-11, murine GDF-11, rat GDF-11, porcine GDF-11, ovine GDF-11, turkeyGDF-11, and baboon GDF-11.

[0049] GDF-8 and GDF-11 inhibitory peptides can be identified andisolated from media of cells expressing GDF-8 or GDF-11 using techniquesknown in the art for purifying peptides or proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for the GDF-8 or GDF-11 inhibitor, or a portionthereof. In one embodiment, the media obtained from cultures of cellswhich express GDF-8 or GDF-11 are subjected to high performance liquidchromatography (HPLC). The samples obtained can then be tested for GDF-8or GDF-11 inhibitory activity as described below.

[0050] Alternatively, GDF-8 and GDF-11 peptide inhibitors can beidentified by screening fragments of GDF-8 or GDF-11 for inhibitoryactivity. GDF-8 or GDF-11 fragments can be produced by a variety of artknown techniques. For example, specific oligopeptides (approximately10-25 amino acids-long) spanning the GDF-8 or GDF-11 sequence can besynthesized (e.g., chemically or recombinantly) and tested for theirability to inhibit GDF-8 or GDF-11, for example, using the assaysdescribed herein. The GDF-8 or GDF-11 peptide fragments can besynthesized using standard techniques such as those described inBodansky, M. Principles of peptide Synthesis, Springer Verlag, Berlin(1993) and Grant, G. A (ed.). Synthetic Peptides: A User's Guide, W. H.Freeman and Company, New York (1992). Automated peptide synthesizers arecommercially available (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600).

[0051] Alternatively, GDF-8 or GDF-11 1 fragments can be produced bydigestion of native or recombinantly produced GDF-8 or GDF-11 by, forexample, using a protease, e.g., trypsin, thermolysin, chymotrypsin, orpepsin. Computer analysis (using commercially available software, e.g.MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used toidentify proteolytic cleavage sites.

[0052] GDF-8 or GDF-11 inhibitors used in the methods of the inventionare preferably isolated. As used herein, an “isolated” or “purified”protein or biologically active peptide thereof is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the GDF-8 or GDF-11 protein or peptide isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of GDF-8 or GDF-11 proteinor peptide thereof in which the protein or peptide thereof is separatedfrom cellular components of the cells from which it is isolated orrecombinantly produced. In one embodiment, the language “substantiallyfree of cellular material” includes preparations of GDF-8 or GDF-11protein or peptide thereof having less than about 30% (by dry weight) ofnon-GDF-8 or GDF-11 protein or peptide thereof (also referred to hereinas a “contaminating protein”), more preferably less than about 20% ofnon-GDF-8 or GDF-11 protein or peptide thereof, still more preferablyless than about 10% of non-GDF-8 or GDF-11 protein or peptide thereof,and most preferably less than about 5% non-GDF-8 or GDF-11 protein orpeptide thereof. When the GDF-8 or GDF-11 protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.

[0053] A two-step method can be used to produce and isolate suchproteolytically cleaved GDF-8 or GDF-11 peptides The first step involvesenzymatic digestion of the GDF-8 or GDF-11 protein. GDF-8 or GDF-11 canbe produced either as a dimer from CHO cell conditioned media, as amonomer in E coli or yeast, or isolated from cells which naturallyproduce GDF-8 or GDF-11. Following purification of GDF-8 or GDF-11monomers or dimers by, for example, HPLC chromatography, their enzymaticdigestion is performed as described infra. The amino acids cleavedduring the digestion depend on the specific protease used in theexperiment as is known in the art. For example, if the protease ofchoice were trypsin, the cleavage sites would be amino acids arginineand lysine. The GDF-8 or GDF-11 protein can be digested using one ormore of such proteases.

[0054] After the digestion, the second step involves the isolation ofpeptide fractions generated by the protein digestion. This can beaccomplished by, for example, high resolution peptide separation asdescribed infra. Once the fractions have been isolated, their GDF-8 orGDF-11 inhibitory activity can be tested for by an appropriate bioassay,as described below.

[0055] The proteolytic or synthetic GDF-8 or GDF-11 fragments cancomprise as many amino acid residues as are necessary to inhibit, e.g.,partially or completely, GDF-8 or GDF-11 function, and preferablycomprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100 or more amino acids in length.

[0056] In one embodiment, peptides are selected which do not contain asufficient number of T cell epitopes to induce T cell mediated immuneresponses and/or which contain a sufficient number of B cell epitopes toelicit antibodies when administered to a mammal. Preferred GDF-8 orGDF-11 peptide inhibitors do not contain a sufficient number of T cellepitopes to induce T-cell mediated (e.g., cytokine) responses. However,B cell epitopes may be desirable and can be selected for by, forexample, testing the peptide's ability to elicit an antibody response,as discussed below.

[0057] T cell epitopes within GDF-8 or GDF-11 fragments can beidentified using a number of well known techniques. For example, T cellepitopes can be predicted using algorithms (see e.g., Rothbard, J. andTaylor, W. R. (1988) EMBO J. 7:93-100; Berzofsky, J. A. (1989) PhilosTrans R. Soc. Lond. 323:535-544). Preferably, human T cell epitopeswithin a GDF-8 or GDF-11 protein can be predicted using known HLA classII binding specific amino acid residues. One algorithm for predictingpeptides having T cell stimulating activity which has been used withsuccess is reported in Rothbard, 1st Forum in Virology, Annals of thePasteur Institute, pp 518-526 (December, 1986), Rothbard and Taylor,(1988) Embo, 7:93-100 and EP 0 304 279. These documents report defininga general T cell pattern (algorithm), its statistical significance andits correlation with known epitopes as well as its successful use inpredicting previously unidentified T cell epitopes of various proteinantigens and autoantigens. The general pattern for a T cell epitope asreported in the above-mentioned documents appears to contain a linearpatter composed of a charged amino acid residue or glycine followed bytwo hydrophobic residues. Other algorithms that have been used topredict T cell epitopes of previously undefined proteins include analgorithm reported by Margalit et al., (1987) J. Immunol.,138:2213-2229, which is based on an amphipathic helix model.

[0058] Other methods for identifying T cell epitopes involve screeningGDF-8 or GDF-1 inhibitory peptides of the invention for human T cellstimulating activity. This can be accomplished using one or more ofseveral different assays. For example, in vitro, T cell stimulatoryactivity can be assayed by contacting a peptide of the invention with anantigen presenting cell which presents appropriate MHC molecules in a Tcell culture. Presentation of a GDF-8 or GDF-11 inhibitory peptide ofthe invention in association with appropriate MHC molecules to T cells,in conjunction with the necessary costimulation can have the effect oftransmitting a signal to the T cell that induces the production ofincreased levels of cytokines, particularly of interleukin-2 andinterleukin-4. The culture supernatant can be obtained and assayed forinterleukin-2 or other known cytokines. For example, any one of severalconventional assays for interleukin-2 can be employed, such as the assaydescribed in Proc. Natl. Acad. Sci USA, 86:1333 (1989) the entirecontents of which are incorporated herein by reference. A kit for anassay for the production of interferon is also available from GenzymeCorporation (Cambridge, Mass.).

[0059] A common assay for T cell proliferation entails measuringtritiated thymidine incorporation. The proliferation of T cells can bemeasured in vitro by determining the amount of ³H-labeled thymidineincorporated into the replicating DNA of cultured cells. Therefore, therate of DNA synthesis and, in turn, the rate of cell division can bequantified.

[0060] Other preferred peptide inhibitors of GDF-8 or GDF-11 are locatedon the surface of the GDF-8 and GDF-11 proteins, e.g., hydrophilicregions, as well as regions with high antigenicity or fragments withhigh surface probability scores can be identified using computeranalysis programs well known to those of skill in the art (Hopp andWood, (1983), Mol.Immunol., 20, 483-9, Kyte and Doolittle, (1982), J.Mol. Biol., 157, 105-32, Corrigan and Huang, (1982), Comput. ProgramsBiomed, 3, 163-8).

[0061] Still other preferred peptides of GDF-8 or GDF-11 to be testedfor GDF-8 or GDF-11 inhibitory activity include one or more B-cellepitopes. Such peptides can be identified by immunizing a mammal withthe peptide, either alone or combined with or linked to an adjuvant(e.g., a hapten), and testing sera from the immunized animal foranti-GDF-8 or GDF-11 antibodies. Preferred peptides generate anti-GDF-8or GDF-11 antibodies which inhibit GDF-8 or GDF-11 activity, indicatingthat these peptides are somehow related to the protein's activity (e.g.,correspond to all or a portion of the active site). For example, serafrom immunized animals can be tested for GDF-8 or GDF-11 inhibitoryactivity using any of the GDF-8 or GDF-11 bioassays described herein.

[0062] Alternatively, anti-GDF-8 or anti-GDF-11 antibodies or antibodyfragments can be administered directly to a subject to inhibit GDF-8 orGDF-11 activity. Preferred antibodies include monoclonal antibodies,including humanized, chimeric and human monoclonals or fragmentsthereof.

[0063] To generate such antibodies, a proteolytic or synthetic GDF-8 orGDF-11 fragment (alone or linked to a suitable carrier or hapten) can beused to immunize a subject (e.g., a mammal including, but not limited toa rabbit, goat, mouse or other mammal). For example, the methodsdescribed in U.S. Pat. Nos. 5,422,110; 5,837,268; 5,708,155; 5,723,129;and 5,849,531, can be used and are incorporated herein by reference. Ina preferred embodiment, the mammal being immunized does not containendogenous GDF-8 or GDF-11 (e.g., a GDF-8 or GDF-11 knock-out transgenicanimal). The immunogenic preparation can further include an adjuvant,such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic proteolytic or synthetic GDF-8 or GDF-11 fragmentpreparation induces a polyclonal anti-GDF-8 or GDF-11 antibody response.The anti-GDF-8 or GDF-11 antibody titer in the immunized subject can bemonitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized GDF-8 or GDF-11.Subsequently, the sera from the immunized subjects can be tested fortheir GDF-8 or GDF-11 inhibitory activity using any of the bioassaysdescribed herein.

[0064] Alternatively, is also possible to immunize subjects (e.g., GDF-8and GDF-11 knockout mice) with plasmids expressing GDF-8 and GDF-11using DNA immunization technology, such as that disclosed in U.S. Pat.No. 5,795,872, Ricigliano et al., “DNA construct for immunization”(1998), and in U.S. Pat. No. 5,643,578, Robinson et al., “Immunizationby inoculation of DNA transcription unit” (1997).

[0065] The antibody molecules directed against GDF-8 or GDF-11 can beisolated from the mammal (e.g., from the blood) and further purified bywell known techniques, such as protein A chromatography to obtain theIgG fraction. At an appropriate time after immunization, e.g., when theanti-GDF-8 or GDF-11 antibody titers are highest, antibody-producingcells can be obtained from the subject and used to prepare e.g.,monoclonal antibodies by standard techniques, such as the hybridomatechnique originally described by Kohler and Milstein (1975) Nature256:495497) (see also, Brown et al. (1981) J. Immunol. 127:53946; Brownet al. (1980) J. Biol Chem .255:4980-83; Yeh et al. (1976) Proc. Natl.Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer29:269-75), the more recent human B cell hybridoma technique (Kozbor etal. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96) or trioma techniques. The technology for producing monoclonalantibody hybridomas is well known (see generally R. H. Kenneth, inMonoclonal Antibodies: A New Dimension In Biological Analyses, PlenumPublishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J.Biol Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet.3:231-36). Briefly, an immortal cell line (typically a myeloma) is fusedto lymphocytes (typically splenocytes) from a mammal immunized with aGDF-8 or GDF-11 immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds GDF-8 or GDF-11.

[0066] Any of the many well known protocols used for fusing lymphocytesand immortalized cell lines can be applied for the purpose of generatingan anti-GDF-8 or GDF-11 monoclonal antibody (see, e.g., G. Galfre et al.(1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line. Preferred immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindGDF-8 or GDF-11 , e.g., using a standard ELISA assay. The antibodies canthen be tested for GDF-8 or GDF-11 inhibitory activity using, forexample, the assays described herein.

[0067] In another aspect of the invention, GDF-8 protein fragmentscomprise all or a portion of the GDF-8 pro-domain. The pro-domain ofTGF-β has been shown to have inhibitory activity against the matureactive TGF-β (Bottinger et. al., (1996) PNAS, 93, 5877-5882; Gentry andNash, (1990) Biochemistry, 29, 6851-6857). Since GDF-8 is a member ofthe TGF-β superfamily, the pro-domain of GDF-8 may also act as aninhibitor to the active GDF-8. The pro-domain of GDF-8 can be generatedby expressing it using various expression systems (e.g. CHO, baculovirusand the like). The expressed pro-domain of GDF-8 can be purified by, forexample, using the method described in Bottinger et. al. (supra) or anyother art recognized method for purifying peptides. Alternatively, thepro-domain can be tagged to, for example, FLAG or 6His, as describedbelow.

[0068] Based on the information obtained for 1TGF-, peptide fragmentsthat span the C-terminus of the mature GDF-8 can be designed andsynthesized. Preferably, the GDF-8 peptide fragments are about 25 aminoacids long. In other preferred embodiments, the GDF-8 peptide fragmentscan have a sequence length of about, 20-25, 25-30, 30-35, 35-40, or40-45 amino acid residues in length. The GDF-8 peptide fragments modeledafter the aforementioned pentacosapeptide can then be tested for GDF-8or GDF-11 inhibitory activity using the assays described herein.

[0069] GDF-8 or GDF-11 inhibitors for use in the methods of the presentinvention can be identified using a variety of appropriate bioassayswhich test for the inhibition of GDF-8 or GDF-11 activity. The abilityof the GDF-8 or GDF-11 inhibitors to inhibit GDF-8 or GDF-11 activity ispreferably specific, i.e., the GDF-8 inhibitor can specifically inhibitthe GDF-8 protein and the GDF-11 inhibitor can specifically inhibit theGDF-11 protein. In certain embodiments, the GDF-8 inhibitor is also ableto inhibit GDF-11 activity and the GDF-11 inhibitor is also able toinhibit GDF-8 activity.

[0070] As used herein, the term “bioassay” includes any assay designedto identify a GDF-8 or GDF-11 inhibitor. The assay can be an in vitro oran in vivo assay suitable for identifying whether a GDF-8 or GDF-11inhibitor can inhibit one or more of the biological functions of GDF-8or GDF-11. Examples of suitable bioassays include DNA replicationassays, transcription-based assays, creatine kinase assays, assays basedon the differentiation of 3T3-L1 pre-adipocytes, assays based on glucoseuptake control in 3T3-L1 adipocytes, and immunological assays (describedin subsection II).

[0071] It has been established that GDF-8 modulates the protein levels,and therefore the activity, of a muscle-specific enzyme, creatinekinase. This effect of GDF-8 or GDF-11 can be used to screen fractionsthat contain potential GDF-8 or GDF-11 inhibitors. This assay can beperformed in the mouse skeletal myoblast cell line C1C12 or in primarychick myoblasts isolated from Day 11 chick embryos. Cells are grown in48-well trays in serum-containing medium that maintains themundifferentiated. When a 70% confluence has been reached, medium isswitched to 1% serum, thus allowing differentiation and creatine kinaseexpression. At the time of the switch, the potential GDF-8 orGDF-11-inhibitory fraction is added to some wells, followed some timelater by GDF-8 or GDF-11 itself Cells are returned to the incubator foran additional two to three day period. In the end, cells are lysed andcreatine kinase activity is measured in the lysates using a commerciallyavailable kit (available by Sigma, St Louis, Mo.).

[0072] Uses

[0073] In one embodiment, the method of the invention can be used eitherin vitro or in vivo to modulate (i.e., upregulate) the expression of ahexose transporter, such as GLUT4 or GLUT1, in a cell which expressesthese transporters, such as a muscle and/or fat cell. This is achievedby inhibiting the activity or expression of GDF-8 or GDF-11 in the cellor outside the cell.

[0074] In another embodiment, the method of the invention can be usedeither in vitro or in vivo to increase insulin sensitivity and/orglucose uptake by a cell.

[0075] In another embodiment, the method of the invention can be used totreat a disease characterized by insufficient GLUT4 expression, insulindysfunction (e.g., resistance, inactivity or deficiency) and/orinsufficient glucose transport into cells. Such diseases include, butare not limited to diabetes, hyperglycemia and obesity.

[0076] In another embodiment, the method of the invention can be used tocreate a novel in vitro model, in which GDF-8 is utilized to examineglucose uptake or glucose metabolism in adipocytes. GDF-8, which isspecifically expressed in muscle and fat in vivo, inhibits 3T3-L1adipocyte differentiation by directly or indirectly suppressing theexpression of adipocyte-specific genes, e.g. the GLUT4 transporter.GDF-8 can, therefore, be used as a prototype regulator of these genes inthe 3T3-L1 cell system. This system can be a model for understanding therole of GDF-8 on the regulation of adipocyte-specific gene expressionand protein activity of molecules such as, but not limited to,transcription factors, signal transduction proteins, leptin, fatty acidbinding protein, fatty acid synthase, peroxisome proliferator-activatedreceptors, uncoupling proteins 1 and 2, and molecules that areactivated, inactivated, or modified by the actions of GDF-8.

[0077] Other uses for the methods of the invention will be apparent toone of ordinary skill in the art from the following Examples and Claims.

[0078] Administration of GDF-8 and GDF-11 Inhibitors in PharmaceuticalCompositions

[0079] GDF-8 and GDF-11 inhibitors used in the methods of the presentinvention are generally administered to a subject in the form of asuitable pharmaceutical composition. Such compositions typically containthe inhibitor and a pharmaceutically acceptable carrier. As used hereinthe language “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the GDF-8 inhibitor, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

[0080] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples ofsuitable routes of administration include parenteral, e.g., intravenous,intradermal, subcutaneous, oral (e.g., inhalation), transdermal(topical), transmucosal, and rectal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

[0081] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0082] Sterile injectable solutions can be prepared by incorporating theGDF-8 inhibitor in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the GDF-8 inhibitor into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0083] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the GDF-8inhibitor can be incorporated with excipients and used in the form oftablets, troches, or capsules. oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0084] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0085] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0086] The GDF-8 inhibitor can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

[0087] In one embodiment, the GDF-8 inhibitors are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

[0088] Methods for preparation of such formulations will be apparent tothose skilled in the art. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

[0089] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0090] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. GDF-8 inhibitors which exhibit large therapeutic indices arepreferred. While GDF-8 inhibitors that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchGDF-8 inhibitors to the site of affected tissue in order to minimizepotential damage to uninfected cells and, thereby, reduce side effects.

[0091] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any GDF-8inhibitor used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test GDF-8 inhibitor which achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybemeasured, for example, by high performance liquid chromatography.

[0092] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0093] The GDF-8 inhibitors of the present invention, e.g., theanti-sense oligonucleotide inhibitors, can further be inserted intovectors and used in gene therapy. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system

[0094] Vectors suitable for use in gene therapy are known in the art.For example, adenovirus-derived vectors can be used. The genome of anadenovirus can be manipulated such that it encodes and expresses a geneproduct of interest but is inactivated in terms of its ability toreplicate in a normal lytic viral life cycle. See for example Berkner etal. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science252:431434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 d1324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known tothose skilled in the art. Recombinant adenoviruses can be advantageousin certain circumstances in that they are not capable of infectingnondividing cells. Furthermore, the virus particle is relatively stableand amenable to purification and concentration, and as above, can bemodified so as to affect the spectrum of infectivity. Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267). Mostreplication-defective adenoviral vectors currently in use and thereforefavored by the present invention are deleted for all or parts of theviral E1 and E3 genes but retain as much as 80% of the adenoviralgenetic material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner etal., supra; and Graham et al. in Methods in Molecular Biology, E. J.Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp.109-127).Expression of the gene of interest comprised in the nucleic acidmolecule can be under control of, for example, the E1A promoter, themajor late promoter (MLP) and associated leader sequences, the E3promoter, or exogenously added promoter sequences.

[0095] Yet another viral vector system useful for delivery of the GDF-8inhibitors of the invention is the adeno-associated virus (AAV).Adeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle. (Fora review see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)158:97-129). Adeno-associated viruses exhibit a high frequency of stableintegration (see for example Flotte et al. (1992) Am. J. Respir. Cell.Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; andMcLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing asfew as 300 base pairs of AAV can be packaged and can integrate. Spacefor exogenous DNA is limited to about 4.5 kb. An AAV vector such as thatdescribed in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can beused to introduce DNA into T cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad Sci. USA 81:6466-6470; Tratschinet al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988)Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619;and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790). Other viralvector systems that may be useful for delivery of the GDF-8 inhibitorsof the invention are derived from herpes virus, vaccinia virus, andseveral RNA viruses.

[0096] The following examples are intended to illustrate but not limitthe invention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXEMPLIFICATION

[0097] Materials and Methods

[0098] The following studies were performed at Intracel (Rockville, Md.)using six week old male Balb/c mice. GDF-8 knockout and wild-type(control) mice were obtained from Dr. S. J. Lee (Johns HopkinsUniversity, See McPherron et al., Nature 387:83-90 (1997)).

[0099] Recombinant human GDF-8 was produced in Chinese Hamster Ovarian(CHO) cells. The secreted protein was purified using several steps ofchromatography to obtain substantially homogenous GDF-8.

[0100] Other materials and methods are described in the Examples below.

EXAMPLE 1 Effect of GDF-8 Knockout on GLUT4 Protein Expression in MuscleCells

[0101] To assess the impact on protein expression of the muscle cellglucose transporter, GLUT4, of knocking out natural GDF-8 expression,samples of various muscles were taken from both wild-type and GDF-8knockout mice. Muscle samples were fixed in 10% (v/v) neutral bufferedformaldehyde (StatLab, Lewisville, Tex.) for 8 hours at room temperaturefollowed by embedding in Paraplast® X-tra tissue-embedding medium(Oxford Labware, St. Louis, Mo.). Cross sections of mouse muscle sampleswere prepared. Slides were preheated in an oven at 60° C. for at least30 min prior to GLUT4 immunodetection. Paraffin sections weredeparaffinzed in xylene three times, 5 min each. Sections wererehydrated and then blocked with 20% normal goat serum (Vector,Burlingame, Calif.) in “Antibody Diluent” (DAKO, Carpinteria, Calif.)for 20 minutes.

[0102] Sections were incubated with rabbit anti-GLUT4 (Alpha DiagnosticInternational, San Antonio, Tex.) diluted in Antibody Diluent at aconcentration of 2 μg/ml overnight at room temperature. Sections wererinsed with OptiMax Wash Buffer (Biogenex, San Ramon, Calif.) andincubated with biotinylated goat anti-rabbit immunoglobulin (BioGenex)for conjugated streptavidin (BioGenex) for 30 min. Sections were rinsedwith the wash buffer and then DAB substrate (DAKO) was applied forvisualizing the antibody binding sites. Sections were counterstainedwith methyl green (DAKO) and mounted using Cytoseal™ 60 (StephensScientific, Riverdale, N.J.). Brown staining indicates the expression ofGLUT4 and green staining identifies the nucleus.

[0103] As shown in FIGS. 1A and 1B, GDF-8 knockout mouse samples displaysignificantly increased GLUT4 expression (as indicated by significantlyincreased staining with anti-GLUT4 antibody) compared to wild-typesamples, regardless of the type of muscle examined. This indicates thatGDF-8 causes a decrease in the expression of GLUT4 in these mice.

EXAMPLE 2 Effect of GDF-8 Administration on GLUT4 Protein Expression inMuscle Cells

[0104] The following study was performed to assess the converse of whatwas found in Example 1, i.e., whether exogenous GDF-8 represses theexpression of GLUT4 in muscle cells (as predicted from Example 1), andalso whether administration of GDF-8 can counteract the effects of theGLUT4 stimulator, insulin.

[0105] Mice were randomized to receive either an intramuscular(gastrcenemius muscle) injection of fifty microliters, containing 5micrograms of recombinant human GDF-8 (in buffer containing 20 mM NaPO₄,150 mM NaCl, 0.1 mg/ml BSA, pH 6.5), or buffer alone. Twenty minuteslater mice received an intraperitoneal injection of either porcineinsulin purchased from Sigma Chemicals, St. Louis, Mo. (13 Units/kg in0.1 ml of the same buffer used above but at pH 7.0), or buffer alone.One hour after insulin administration the animals were sacrificed andsamples of the injected muscle were removed.

[0106] As shown in FIG. 2, the administration of exogenous GDF-8 aloneresults in a significant decrease in GLUT4 expression in mousegastrocnemius cells. In contrast, the administration of insulin aloneresults in the opposite effect—a significant increase in GLUT4expression (i.e., staining) is observed in these cells. When GDF-8 andinsulin are simultaneously administered, the GLUT4 staining patternappears close to that of untreated control cells, suggesting that thesetwo molecules have opposite regulatory effects on GLUT4.

EXAMPLE 3 Effect of GDF-8-Expressing CHO Cell Tumors in Nude Mice

[0107] The results from the preceding Examples indicate that GDF-8 playsan important role in the regulation of GLUT4 protein expression inmuscle cells. To further examine the role of GDF-8 in the regulation ofoverall glucose metabolism in vivo, a Chinese Hamster Ovarian (CHO)tumor cell line producing human GDF-8 (hGDF-8) was injected into nudemice to form a tumor expressing GDF-8. This CHO tumor cell injectionapproach has been used as a model for determining the effects of variousgene products in vivo (Black et al., Endocrinology 123:2657-2659(1991)).

[0108] CHO cells expressing hGDF-8 were cultured in alpha medium with0.1 micromolar methotrexate and 1 mg/ml G418, while the control CHOcells (containing an empty expression vector) were cultured in alphamedium with 0.1 micromolar methotrexate. The cells were harvested bytrypsinization and resuspended in PBS at a concentration of 2×10⁷cells/ml. A subcutaneous injection of 1×10⁷ cells in 0.5 ml was madeinto the right thigh of male nu/nu NCR mice. Body weight and tumor sizeswere measured twice a week for the duration of the experiment. Northernblot analysis of mRNA isolated from the CHO GDF-8 tumors confirmed thatGDF-8 was expressed.

[0109] The systemic effects of the GDF-8 produced by the developing CHOGDF-8 tumor were assessed. As shown in FIG. 3, the CHO tumorsoverexpressing GDF-8 caused dramatic total body weight loss (a decreaseof 25%) within 20 days in the nude mice, compared to their weight at theonset of the experiment. In contrast, the mice harboring control CHOtumors not expressing GDF-8 had a slight weight gain (FIG. 3).

[0110] As shown in FIG. 4, the CHO GDF-8 tumor-bearing mice showed aneven more dramatic weight loss (35%) when the net body weights(total-tumor) were compared with that of control tumor-bearing mice(FIG. 4, Panel A). The weight loss was not due to the size of the CHOGDF-8 tumor, since control tumor weight was actually heavier than CHOGDF-8 tumor (FIG. 4, Panel B, and FIG. 5).

[0111] Individual tissues from CHO and CHO GDF-8-expressingtumor-bearing animals were also isolated and weighed. Muscles and fatpads from CHO GDF-8 tumor-bearing animals showed a significant decreasein weight compared to CHO tumor-bearing animals (FIG. 4, Panels C,D, andE). This general wasting and reduction in skeletal muscle massdemonstrates that the GDF-8 protein produced from implanted CHO cellsacts in a manner strictly compatible with, and as expected from, theresults of the GDF-8 knock-out approach.

[0112] To assess whether GDF-8 is involved in systemic glucose handling,wild-type nude mice carrying CHO-GDF-8 tumors were tested for elevatedglucose. As shown in FIG. 6, when compared to control CHO tumor-bearingmice, CHO GDF-8 tumor-bearing animals exhibited hyperglycemia and asignificant decrease in GLUT4 levels in muscle tissues. Taken togetherwith the fact that the GDF-8 knockout mice were hypoglycemic and hadincreased GLUT4 expression levels in muscle, these results suggestedthat GDF-8 increases glucose levels in the serum by inhibiting GLUT4levels in vivo.

EXAMPLE 4 Systemic Effects of GDF-8 Knockout in Mice

[0113] Transgenic mice in which the GDF-8 gene is knocked out hadcharacteristic systemic problems, particularly hypoglycemia, significantmuscle hypertrophy, and a dramatic decrease in overall body fat. Thesefindings indicate not only that the modulation of GDF-8 may enable theregulation of glucose levels in the serum, thus serving as a treatmentfor diabetes, but also that GDF-8 may be useful in treating obesity andother disorders related thereto.

[0114] While GDF-8 knock-out mice provide a model for postulating thegeneral role of GDF-8 in regulating muscle and fat growth and metabolicfunction, it is unclear whether the observed changes are a consequenceof embryonic GDF-8 deficiency or the result of post-natal development.Thus, this Example, as well as the immediately preceding Example (i.e.,Example 3) demonstrate for the first time that GDF-8 has an importantphysiological role in the adult animal. These two examples provideunambiguous support to the concept that modulating GDF-8 expression andactivity post-natally is a means of regulating muscle and fat growth andmetabolic function including, but not limited to, muscle growth, glucosehomeostasis and diabetes susceptibility.

EXAMPLE 5 Effect of GDF-8 on the Differentiation of 3T3-L1Pre-Adipocytes

[0115] To better characterize the effects of GDF-8 on glucosehomeostasis, 3T3-L1 cells were utilized as a model for adipocytes, acell type acutely responsive to insulin through its ability to increasehexose transport through GLUT4. These cells have been well characterizedas an excellent model for adipogenesis (Hwang et al., Annu. Rev. CellDev. Biol. 13, 231-259 (1997), and MacDougald and Lane, Annu. Rev.Biochem. 64, 345-373 (1995)). When these cells are stimulated withinsulin, dexamethasone and isobutylmethylxanthine (IBMX), they areinduced to undergo both morphological and biochemical changes resultingin their differentiation into adipocytes.

[0116] When undifferentiated pre-adipocytes reached confluence,differentiation was predictably achieved (Spiegelman et al., J. Biol.Chem. 268:6823-6826 (1993)) by successive replacements of theirserum-containing DMEM media as follows:DMEM+serum+IBMX+dexamethasone+insulin for 2 days, DMEM+serum+insulin for2 additional days. After this, the media was again replaced withDMEM+/−serum. GDF-8 and other growth factors were added at the onset ofdifferentiation and were resupplied at each additional medium change.Adipocytes were maintained for an additional 3 to 5 days in this mediafor full differentiation to take place.

[0117] As shown in FIG. 7, GDF-8 inhibited differentiation of these3T3-L1 pre-adipocytes to adipocyte cells. The addition of GDF-8 to3T3-L1 cells at the onset of induction to differentiate into adipocytesprevented the conversion of pre-adipocytes to adipocytes, as seen by themaintenance of pre-adipocyte morphology and the near-absence ofrefractile cells that contain lipid droplets (FIG. 7). In addition, asshown in FIG. 8, at the RNA level, GDF-8 inhibited the expression ofGLUT4 mRNA, a known adipocyte marker.

[0118]FIG. 7 also shows that GDF-8 is able to mimic the effects of bothTNF-α and TGF-β₁ on GLUT4 mRNA levels. Importantly, GLUT4 is known to bethe key molecule responsible for insulin-sensitive glucose transport notonly in muscle tissue, but also in fat cells. Thus GDF-8 plays a role inmediating insulin resistance associated with Type II diabetes. GDF-8,which is specifically expressed in the muscle and in fat, can fullymimic the previously established effects of two broadly-expressedcytokines, namely TNF-α and TGF-β, on adipocyte differentiation andmetabolism (Szalkowski et al., Endocrinology 136:1474-1481 (1995)). Dueto its specific expression, GDF-8 may be, among the three, thephysiologically most relevant polypeptide that regulates such processesin vivo.

EXAMPLE 6 Effect of GDF-8 on Glucose Uptake in 3T3-L1 Adipocytes

[0119] In differentiated adipocytes, insulin stimulates glucosetransport through the GLUT4 transporter in a dose-dependent fashion.Thus, the ability of GDF-8 to interfere with this insulin-dependentglucose uptake mechanism was examined as follows.

[0120] Upon completion of differentiation, a glucose transport assay wasperformed on 3T3-L1 cells. GDF-8 was added in the final 72 hours ofdifferentiation. Insulin was added in Krebs-Ringer solution for 20 min.,followed by addition of [³H]-deoxyglucose (1 mCi/ml) for 10 min. Afterextensive washing, cells were lysed with Triton X-100 and thecell-associated radioactive glucose (due to GLUT4-mediated uptake) wasdetermined by scintillation counting.

[0121] As shown in FIG. 9, GDF-8 reduced the insulin-sensitivity ofthese cells, as measured by fold-induction of glucose uptake, in adose-dependent manner. This reduced insulin-sensitivity of glucosetransport correlated with the decrease of GLUT4 mRNA levels in thesecells after GDF-8 treatment (FIG. 8). Thus, this assay offers an invitro correlate of GDF-8 activity that may be relevant to its in vivoeffects on body fat and muscle metabolic functions.

[0122] Besides a decrease in the GLUT4 mRNA levels (seen in FIG. 8), anadditional important observation was made that in the 3T3-L1 cells,GDF-8 actually increased basal glucose transport by about 50%. Thisincrease in baseline should-also contribute to the reducedfold-increases in glucose uptake in response to insulin. Since thisbasal transport is mainly effected by the ubiquitous GLUT1 transporter(another hexose transporter), it indicates that the insulininsensitivity observed after GDF-8 treatment in adipocytes can stem froma combination of an increase in basal transport (through GLUT1 and otherglucose transporters) and a concomitant decrease in insulin-stimulatedtransport (through GLUT4). However, the increase in basal level ofglucose is not limited to the effect of GLUT1. Additional glucosetransporters can also increase the basal level of glucose in the 3T3-L1cells.

EXAMPLE 7 Effect of GDF-8 in Diabetes Disease Models

[0123] The foregoing Examples demonstrate that GDF-8 inhibition canincrease GLUT4 transcription and expression, and thereby restore insulinsensitivity and reduce systemic glucose levels in a subject. Theforegoing Examples further demonstrate that GDF-8 inhibition upregulatesdifferentiation of adipocytes, and thereby increases insulin-sensitiveglucose uptake.

[0124] Together, this data suggests that interfering with GDF-8 functioncould have important applications for the treatment of Type II diabetes,obesity and disorders related to obesity. To pursue these potentialapplications, the following approaches can be taken.

[0125] A. Analysis of the Effect of the GDF-8 Mutation in Mouse Modelsof Obesity/Diabetes

[0126] GDF-8 knockout mice can be crossed with various mouse strainsexhibiting obesity, particularly ob/ob, db/db, and mice carrying thelethal yellow mutation. Serum levels of known molecular markers ofobesity, such as glucose, insulin, lipids, and creatine kinase aremonitored and compared to control animals lacking the GDF-8 knockout, asan indication of the presence of this condition in the test animals.Functional assays for diabetes/obesity including, but not limited to, aninsulin sensitivity assay, a glucose tolerance assay and an ex-vivoglucose uptake by isolated muscle assay also can be performed to monitorthe effect of GDF-8 knockout on progeny mice.

[0127] In progeny mice carrying both the GDF-8 knockout and therecessive obesity genotype, the lack of GDF-8 should suppress theobesity phenotype as compared to that of corresponding control mice, asmeasured by serum levels of known molecular markers of obesity, such asglucose, insulin, lipids and creatine kinase. Additionally, thedevelopment of diabetes in these animals should be delayed or preventedby the absence of GDF-8.

[0128] B. Analysis of GDF-8 Knockout Mice in Various Models of Diabetes

[0129] GDF-8 knockout mice can be subjected to agents capable ofinducing experimental diabetes, such as streptozotocin. An analysis ofserum levels of molecular diabetes markers, such as glucose, insulin,lipids, and creatine kinase is performed in these animals, and comparedto, e.g., streptozotocin-treated wild-type control animals. Functionalassays for diabetes/obesity including, but limited to, an insulinsensitivity assay, a glucose tolerance assay and an ex-vivo glucoseuptake by isolated muscle assay can be performed to monitor the effectof streptozotocin on the treated and non-treated animals. The GDF-8knockout mice should be relatively resistant to such treatments, and theonset of experimental diabetes should be altogether prevented, delayed,or be less severe.

[0130] C. Demonstration of the Efficacy of GDF-8 Inhibitors in MouseModels of Obesity/Diabetes

[0131] Mice serving as models for obesity or diabetes can be treatedwith GDF-8 inhibitors to determine whether inhibition of GDF-8 and thecorresponding impact on GLUT4 ameliorates the symptoms of either obesityor diabetes in these animals.

[0132] Mice with either obesity or diabetes are treated with one or moreGDF-8 inhibitors in a therapeutically effective dose. GDF-8 levels intreated and control mice can be assessed by Western blot analysis usingantibodies specific for GDF-8. Levels of molecules characteristic forobesity and diabetes, such as glucose, insulin, lipids, and creatinekinase can be assessed in serum samples taken from treated and controlanimals. Functional assays for diabetes/obesity including, but notlimited to, an insulin sensitivity assay, a glucose tolerance assay andan ex-vivo glucose uptake by isolated muscle cell assay can be performedto monitor the effect of the inhibitor on treated and non-treatedanimals. Similarly, muscle and fat cell differentiation can be observedin these animals. Analysis of such studies should enable a determinationof the overall effect of the inhibition of GDF-8 on the progression ofdiabetes or obesity in animal models for these diseases.

[0133] All patents, published patent applications and other publishedreferences disclosed herein are hereby expressly incorporated herein intheir entireties by reference.

[0134] Equivalents

[0135] Those skilled in the art will recognize, or will be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

We claim:
 1. A method of increasing expression of GLUT4 in a subjectcomprising administering to the subject a GDF-8 inhibitor.
 2. A methodof increasing insulin sensitivity and glucose uptake by cells in asubject comprising administering to the subject a GDF-8 inhibitor.
 3. Amethod of treating diabetes in a subject comprising administering to thesubject a GDF-8 inhibitor.
 4. The method of any one of claims 1-3,wherein the GDF-8 inhibitor is an antibody or antibody fragment.
 5. Themethod of any one of claims 1-3, wherein the GDF-8 inhibitor is selectedfrom the group consisting of a peptide fragment of GDF-8, adominant-negative mutant of GDF-8, a GDF-8 receptor antagonist, anon-GDF-8 peptide, an antisense nucleic acid and a ribozyme.
 6. Themethod of any one of claims 1-3, wherein the GDF-8 inhibitor is derivedfrom mature GDF-8 protein.
 7. The method of any one of claims 1-3,wherein the GDF-8 inhibitor is derived from the Pro domain of a GDF-8protein.
 8. The method of claim 2, wherein said insulin sensitivity andglucose uptake is increased by modulating the expression of a hexosetransporter selected from the group consisting of GLUT4 and GLUT1. 9.The method of claim 2, wherein the cell is a muscle cell or a precursorthereof.
 10. The method of claim 2, wherein the cell is an adipocyte ora precursor thereof.
 11. The method of claim 3, wherein the subject issuffering from type II diabetes.